Method development for studying bacterial messenger RNA as biomarkers

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Rimaben A. Goswami (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Norman Chiu

Abstract: The gut microbiome is a collection of microbes that exist in the digestive tract, which includes bacteria, viruses and archaea. These microbes are symbiotic to their host and are essential to the proper development and ongoing health of digestive and immune systems. Any fluctuations in the environment can dramatically affect this relationship, resulting in changes of gut microbiome and human health, such as diabetes, asthma, bowel diseases, cancers and obesity. To explore the roles of the gut microbiome in the development and/or prevention of diseases, the profiling of gut microbiome and their activities must be determined. Messenger RNA (mRNA) is a large group of RNA molecules that would be translated into functional proteins molecules, which then perform all sorts of biological activities and structures. In the past, mRNA have been used as biomarkers for monitoring cellular activities, but the use of metatranscriptome to study the gut microbiome activities is limited. For this reason, we set out to explore the use of mRNA as biomarker for monitoring the gut microbiome activities.In this study, we build a simple gut microbe model aiming to demonstrate the relationship between the level of mRNA and gut microbe activities. L. reuteri, a probiotic Gram-positive bacteria was chosen as our model. In order to carry out the initial studies with the selected model, there are a number of technical issues that ought to be addressed. In the first case, due to the lack of 3' poly-A tail in the bacterial mRNA and the limited number of reports on the extraction of RNA from Gram positive bacterial cells, we had compared and evaluated various methods for extracting total RNA from bacterial cells. The goal was to establish a reproducible method that would provide sufficient purity and yield of total RNA. We used Triton X-100 Boiling method for the extraction of total RNA from L. reuteri cells after comparing various methods. The total RNA extracted was usually less than 900 ng/µL in quantity which reduces our choice of using the spectrometer to measure the concentration and purity of our sample. The concentration and purity was measured by the use of NanoDrop 1000 spectrophotometer, which uses about 1-2 µL of sample. The technique used for studying mRNA was gel electrophoresis. Sequencing is another method available to identify presence of a particular mRNA, but there is yet some information required to build a library that will allow us to match a single mRNA to its parent bacterial strain. As this being our first step for the feasibility study for mRNA being used as biomarkers, it’s important to look for a cheaper, easier and quicker method. When it comes to cheap and easy, agarose gel electrophoresis is a well-established method available in the science world. Total RNA were extracted from the bacterial cells and gel electrophoresis was performed on it to explore the changes in mRNA due to a given growth conditions. Since there wasn’t an effective gel electrophoresis method available to inspect all the mRNA from the cell, re-extraction of mRNA smear from an agarose gel was done. With this developed method to monitor mRNA changes in L. reuteri cells, we can get insightful information about the functionality of gut microbe.

Additional Information

Language: English
Date: 2017
Electrophoresis, mRNA, rRNA
Gastrointestinal system $x Microbiology
Messenger RNA
Biochemical markers

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